Egg Extracellular Matrix Research Articles

Shrimp thrombospondin (TSP): presence of O-β1,4 N-acetylglucosamine polymers and its function in TSP chain association in egg extracellular matrix

We characterized the existence of O-β(1,4)-GlcNAc polymers (β1,4GNP) that were anchored on the O-linked glycosylation sites of shrimp thrombospondin (pmTSP-II). There were five putative β1,4GNP linkages on the epithelial growth factor-like domain of pmTSP-II. Antibody against O-β-GlcNAc (CTD110.6) was used to prove the existence of linear and complex β1,4GNP. The antibody well reacted with linear chito-triose, -tetraose and -pentaose conjugated with phosphatidylethanolamine lipid. The immunoreactivity could also be detected with a complex β1,4GNP within pmTSP-II (at MW > 250 kDa). Upon denaturing the protein with SDS-PAGE buffer, the size of pmTSP-II was shifted to be 250 kDa, approximately 2.5 folds larger than the deduced molecular mass of pmTSP-II (110 kDa), suggesting additional association of pmTSP-II apart from its known disulfide bridging. This was confirmed by chitinase digestion on pmTSP-II protein leading to the subsequent smaller protein bands at 110–170 kDa in time- and concentration-dependent manners. These bands well reacted with CTD110.6 antibody and disappeared after extensive chitinase hydrolysis. Together, we believe that β1,4GNP on pmTSP-II serve the function in an inter-chain association to provide structural architecture of egg extracellular matrix, a novel function of pmTSP-II in reproductive biology.

Arabinogalactan proteins and arabinan pectins abound in the specialized matrices surrounding female gametes of the fern Ceratopteris richardii.

Both male and female gametes of archegoniates are highly specialized cells surrounded by an extraprotoplasmic matrix rich in AGPs, which are speculated to facilitate development and gamete fusion through Ca 2+) oscillations. An additional layer, the egg envelope, forms around the egg periphery, except at the fertilization pore, and contains arabinose-rich polymers that presumably impart flexibility for the rapidly growing zygote and embryo. The abundant AGPs and arabinan pectins associated with the eggs of C. richardii not only are integral to development, fertilization, and early embryogenesis, but also may be involved in desiccation tolerance important to the survival of the reproductive gametophyte. A defining feature of gametogenesis in archegoniates is the deposition of a special matrix outside of the plasmalemma of both egg and sperm cells that displaces the primary cell wall away from the protoplasm. It is within this matrix that gamete differentiation occurs. In leptosporangiate ferns, maturation of the egg cell involves the deposition of a second specialized wall, the so-called egg envelope that surrounds the cell except at the fertilization pore, a narrow site where gamete fusion takes place. We provide the first conclusive evidence of the macromolecular constituents in the unique structures surrounding fern egg cells before and after fertilization. To test the hypotheses that the egg extracellular matrix contains arabinogalactan proteins (AGPs) as does the sperm cell matrix, and that cell wall polysaccharides, especially pectins, are components of the egg envelope, we examined the expression patterns of AGPs and cell wall constituents during oogenesis in Ceratopteris richardii. Utilizing histochemical stains for callose, cellulose and AGPs coupled with immunogold localizations employing a suite of monoclonal antibodies to cell wall components (JIM13, JIM8, LM2, LM5, LM6, LM19, LM20 and anticallose), we demonstrate that AGPs, but not pectins, are abundant in the matrix around egg cells and degrading neck canal and ventral canal cells during archegonial development. A striking finding is that both AGPs and (1,5)-α-L-arabinan pectin epitopes are principle components of the egg envelope before and after fertilization, suggesting that they are important in both egg maturation and gamete fusion.

Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins.

The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm–ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm–ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm–ZP binding step.

Glycopattern analysis and structure of the egg extra-cellular matrix in the Apennine yellow-bellied toad, <i>Bombina pachypus (Anura: Bombinatoridae)</i>

We studied the glycopatterns and ultrastructure of the extra-cellular matrix (ECM) of the egg of the Apennine yellow-bellied toad Bombina pachypus, by light and electron microscopy in order to determine structure, chemical composition and function. Histochemical techniques in light microscopy included PAS and Alcian Blue pH 2.5 and 1.0, performed also after β-elimination. Lectin-binding was tested with nine lectins (AAA, ConA, DBA, HPA, LTA, PNA, SBA, UEA-I, WGA). An inner fertilization envelope (FE) and five jelly layers (J1-J5) were observed, differing in histochemical staining, lectin binding and ultrastructure. Most glycans were O-linked, with many glucosamylated and fucosylated residues. The fertilization envelope presented a perivitelline space and a fertilization layer, with mostly neutral glycans. The jelly layers consisted of fibers and granules, whose number and orientation differed between layers. Fibers were densely packed in J(1) and J(4) layers, whereas a looser arrangement was observed in the other layers. Jelly-layer glycans were mostly acidic and particularly abundant in the J(1) and J(4) layers. In the J(1), J(2) and J(5) layers, neutral, N-linked glycans were also observed. Mannosylated and/or glucosylated as well as galactosyl/galactosaminylated residues were more abundant in the outer layers. Many microorganisms were observed in the J(5) layer. We believe that, apart from their functions in the fertilization process, acidic and fucosylated glycans could act as a barrier against pathogen penetration.

Crystal structure of the ZP-N domain of ZP3 reveals the core fold of animal egg coats

Species-specific recognition between the egg extracellular matrix (zona pellucida) and sperm is the first, crucial step of mammalian fertilization. Zona pellucida filament components ZP3 and ZP2 act as sperm receptors, and mice lacking either of the corresponding genes produce oocytes without a zona pellucida and are completely infertile. Like their counterparts in the vitelline envelope of non-mammalian eggs and many other secreted eukaryotic proteins, zona pellucida subunits polymerize using a 'zona pellucida (ZP) domain' module, whose conserved amino-terminal part (ZP-N) was suggested to constitute a domain of its own. No atomic structure has been reported for ZP domain proteins, and there is no structural information on any conserved vertebrate protein that is essential for fertilization and directly involved in egg-sperm binding. Here we describe the 2.3 ångström (A) resolution structure of the ZP-N fragment of mouse primary sperm receptor ZP3. The ZP-N fold defines a new immunoglobulin superfamily subtype with a beta-sheet extension characterized by an E' strand and an invariant tyrosine residue implicated in polymerization. The structure strongly supports the presence of ZP-N repeats within the N-terminal region of ZP2 and other vertebrate zona pellucida/vitelline envelope proteins, with implications for overall egg coat architecture, the post-fertilization block to polyspermy and speciation. Moreover, it provides an important framework for understanding human diseases caused by mutations in ZP domain proteins and developing new methods of non-hormonal contraception.

Renovation of the egg extracellular matrix at fertilization

Extracellular matrices are essential for cell survival and function. This is especially relevant for eggs, which establish a physical barrier at fertilization to protect a new embryo from additional sperm and pathogens. Formation of an extracellular matrix is most dramatic in sea urchins, in which fertilization was first observed in animals with the "sudden appearance of a perfectly transparent envelope" (A. Derbès, 1847). The process of assembling this extracellular "envelope" has been a topic of intense study ever since. Here we integrate the cellular and molecular events necessary to form this fertilization envelope within the first few minutes of a new embryo's life.

Free-radical crosslinking of specific proteins alters the function of the egg extracellular matrix at fertilization

All animal embryos begin development by modifying the egg extracellular matrix. This protein-rich matrix protects against polyspermy, microbes and mechanical stress via enzyme-dependent transformations that alter the organization of its constituents. Using the sea urchin fertilization envelope, a well-defined extracellular structure formed within minutes of fertilization, we examine the mechanisms whereby limited permeability is established within this matrix. We find that the fertilization envelope acquires a barrier filtration of 40,000 daltons within minutes of insemination via a peroxidase-dependent mechanism, with dynamics that parallel requisite production of hydrogen peroxide by the zygote. To identify the molecular targets of this free-radical modification, we developed an in vivo technique to label and isolate the modified matrix components for mass spectrometry. This method revealed that four of the six major extracellular matrix components are selectively crosslinked, discriminating even sibling proteins from the same gene. Thus, specific free-radical chemistry is essential for establishing the embryonic microenvironment of early development.

Complexin I is required for mammalian sperm acrosomal exocytosis

Regulated exocytosis in many cells is controlled by the SNARE complex, whose core includes three proteins that promote membrane fusion. Complexins I and II are highly related cytosolic proteins that bind tightly to the assembled SNARE complex and regulate neuronal exocytosis. Like somatic cells, sperm undergo regulated exocytosis; however, sperm release a single large vesicle, the acrosome, whose release has different characteristics than neuronal exocytosis. Acrosomal release is triggered upon sperm adhesion to the mammalian egg extracellular matrix (zona pellucida) to allow penetration of the egg coat. Membrane fusion occurs at multiple points within the acrosome but how fusion is activated and the formation and progression of fusion points is synchronized is unclear. We show that complexins I and II are found in acrosome-intact mature sperm, bind to SNARE complex proteins, and are not detected in sperm after acrosomal exocytosis (acrosome reaction). Although complexin-I-deficient sperm acrosome-react in response to calcium ionophore, they do not acrosome-react in response to egg zona pellucida proteins and have reduced fertilizing ability, in vitro. Complexin II is present in the complexin-I-deficient sperm and its expression is increased in complexin-I-deficient testes. Therefore, complexin I functions in exocytosis in two related but morphologically distinct secretory processes. Sperm are unusual because they express both complexins I and II but have a unique and specific requirement for complexin I.

Identification of an ADAM2-ADAM3 Complex on the Surface of Mouse Testicular Germ Cells and Cauda Epididymal Sperm

Male mice lacking ADAM2 (fertilin beta) or ADAM3 (cyritestin) are infertile; cauda epididymal sperm (mature sperm) from these mutant mice cannot bind to the egg zona pellucida. ADAM3 is barely present in Adam2-null sperm, despite normal levels of this protein in Adam2-null testicular germ cells (TGCs; sperm precursor cells). Here, we have explored the molecular basis for the loss of ADAM3 in Adam2-null TGCs to clarify the biosynthetic and functional linkage of ADAM2 and ADAM3. A small portion of total ADAM3 was found present on the surface of wild-type and Adam2(-/-) TGCs at similar levels. In the Adam2-null TGCs, however, surface-localized ADAM3 exhibited an increased amount of an endoglycosidase H-resistant form that may be related to instability of ADAM3. Moreover, we found a complex between ADAM2 and ADAM3 on the surface of TGCs and sperm. The intracellular chaperone calnexin was a component of the testicular ADAM2-ADAM3 complex. Our findings suggest that the association with ADAM2 is a key element for stability of ADAM3 in epididymal sperm. The presence of the ADAM2-ADAM3 complex in sperm also suggests a potential role of ADAM2 with ADAM3 in sperm binding to the egg zona pellucida.

Phosphoinositide-dependent pathways in mouse sperm are regulated by egg ZP3 and drive the acrosome reaction

Sperm of many animals must complete an exocytotic event, the acrosome reaction, in order to fuse with eggs. In mammals, acrosome reactions are triggered during sperm contact with the egg extracellular matrix, or zona pellucida, by the matrix glycoprotein ZP3. Here, we show that ZP3 stimulates production of phosphatidylinositol-(3,4,5)-triphosphate in sperm membranes. Phosphatidylinositol-3-kinase antagonists that prevent acrosome reactions and fertilization in vitro, while generation of this phosphoinositide in the absence of ZP3 triggered acrosome reactions. Downstream effectors of phosphatidylinositol-(3,4,5)-triphosphate in sperm include the protein kinases, Akt and PKCζ. These studies outline a signal transduction pathway that plays an essential role in the early events of mammalian fertilization.

Rendezvin: An Essential Gene Encoding Independent, Differentially Secreted Egg Proteins That Organize the Fertilization Envelope Proteome after Self-Association

Preventing polyspermy during animal fertilization relies on modifications to the egg's extracellular matrix. On fertilization in sea urchins, the contents of cortical granules are secreted and rapidly assemble into the egg's extracellular vitelline layer, forming the fertilization envelope, a proteinaceous structure that protects the zygote from subsequent sperm. Here, we document rendezvin, a gene whose transcript is differentially spliced to yield proteins destined for either cortical granules or the vitelline layer. These distinctly trafficked variants reunite after cortical granule secretion at fertilization. Together, they help coordinate assembly of the functional fertilization envelope, whose proteome is now defined in full.

Proteomic analysis of sperm regions that mediate sperm-egg interactions

The sperm interacts with three oocyte-associated structures during fertilization: the cumulus cell layer surrounding the oocyte, the egg extracellular matrix (the zona pellucida), and the oocyte plasma membrane. Each of these interactions is mediated by the sperm head, probably through proteins both on the sperm surface and within the acrosome, a specialized secretory granule. In this study, we have used subcellular fractionation in order to generate a proteome of the sperm head subcellular compartments that interact with oocytes. Of the proteins we identified for which a gene knockout has been tested, a third have been shown to be essential for efficient reproduction in vivo. Many of the other presently untested proteins are likely to have a similarly important role. Twenty-five percent of the cell surface fraction proteins are previously uncharacterized. We have shown that at least two of these novel proteins are localized to the sperm head. In summary, we have identified over 100 proteins that are expressed on mature sperm at the site of sperm-oocyte interactions.

Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway

Epac, a guanine nucleotide exchange factor for the small GTPase Rap, binds to and is activated by the second messenger cAMP. In sperm, there are a number of signaling pathways required to achieve egg-fertilizing ability that depend upon an intracellular rise of cAMP. Most of these processes were thought to be mediated by cAMP-dependent protein kinases. Here we report a new dependence for the cAMP-induced acrosome reaction involving Epac. The acrosome reaction is a specialized type of regulated exocytosis leading to a massive fusion between the outer acrosomal and the plasma membranes of sperm cells. Ca2+ is the archetypical trigger of regulated exocytosis, and we show here that its effects on acrosomal release are fully mediated by cAMP. Ca2+ failed to trigger acrosomal exocytosis when intracellular cAMP was depleted by an exogenously added phosphodiesterase or when Epac was sequestered by specific blocking antibodies. The nondiscriminating dibutyryl-cAMP and the Epac-selective 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate analogues triggered the acrosome reaction in the effective absence of extracellular Ca2+. This indicates that cAMP, via Epac activation, has the ability to drive the whole cascade of events necessary to bring exocytosis to completion, including tethering and docking of the acrosome to the plasma membrane, priming of the fusion machinery, mobilization of intravesicular Ca2+, and ultimately, bilayer mixing and fusion. cAMP-elicited exocytosis was sensitive to anti-alpha-SNAP, anti-NSF, and anti-Rab3A antibodies, to intra-acrosomal Ca2+ chelators, and to botulinum toxins but was resistant to cAMP-dependent protein kinase blockers. These experiments thus identify Epac in human sperm and evince its indispensable role downstream of Ca2+ in exocytosis.

Structural Conservation of Mouse and Rat Zona Pellucida Glycoproteins. Probing the Native Rat Zona Pellucida Proteome by Mass Spectrometry

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.

CASK is in the mammalian sperm head and is processed during epididymal maturation.

Upon adhesion to the zona pellucida or egg extracellular matrix, sperm undergo regulated exocytosis of the acrosomal vesicle. CASK is an adaptor protein that has been implicated in coupling neuronal cell adhesion to regulated exocytosis. In neurons, this scaffolding molecule is associated with several types of transmembrane receptor complexes and connects cell adhesion molecules with ion channels, the actin cytoskeleton, and the cell's exocytotic machinery. We hypothesized CASK might also be an important link between zona pellucida binding and the sperm acrosome reaction. RT-PCR experiments indicated CASK is transcribed in mouse testis. The full size (120 kDa) CASK protein was present in testis from mouse and pig. Immunoblots of mature porcine and murine sperm revealed that the 120 kDa molecule was much less abundant than in testis but the antibody also recognized a group of smaller proteins migrating at 55-65 kDa. Immunofluorescence experiments indicated both the full length and smaller CASK immunoreactive products were found only in the acrosomal region of spermatids and mature sperm and not in other testicular cell types. CASK immunofluorescence was lost following the acrosome reaction. During epididymal maturation, the abundance of the full size CASK decreased and the CASK fragments increased. These results suggest that CASK may be proteolytically processed during epididymal maturation. Because sperm acquire the ability to bind the zona pellucida, acrosome react, and fertilize eggs during epididymal maturation, CASK processing may play a role in the acquisition of these functions.

Major components of a sea urchin block to polyspermy are structurally and functionally conserved

One sperm fusing with one egg is requisite for successful fertilization; additional sperm fusions are lethal to the embryo. Because sperm usually outnumber eggs, evolution has selected for mechanisms that prevent this polyspermy by immediately modifying the egg extracellular matrix. We focus here on the contribution of cortical granule contents in the sea urchin block to polyspermy to begin to understand how well this process is conserved. We identified each of the major constituents of the fertilization envelope in two species of seaurchins, Strongylocentrotus purpuratus and Lytechinus variegatus, that diverged 30 to 50 million years ago. Our results show that the five major structural components of the fertilization envelope, derived from the egg cortical granules, are semiconserved. Most of these orthologs share sequence identity and encode multiple low-density lipoprotein receptor type A repeats or CUB domains but at least two contain radically different carboxy-terminal repeats. Using a new association assay, we also show that these major structural components are functionally conserved during fertilization envelope construction. Thus, it seems that this population of female reproductive proteins has retained functional motifs while gaining significant sequence diversity-two opposing paths that may reflect cooperativity among the proteins that compose the fertilization envelope.

Cross-fertilization and structural comparison of egg extracellular matrix glycoproteins from Xenopus laevis and Xenopus tropicalis

While the anuran amphibian Xenopus laevis is a widely used vertebrate model system, it is not optimal for genetic manipulations due to its tetraploid genome and long generation time. A current alternative amphibian model system, Xenopus tropicalis, has the advantages of a diploid genome and a much shorter generation time. We undertook a comparative investigation of X. tropicalis egg extracellular matrix glycoproteins in relation to those already characterized in X. laevis. Fertilization methods and isolation of egg extracellular molecules were directly transferable from X. laevis to X. tropicalis. Cross-fertilizations were successful in both directions, indicating similar molecules involved in sperm–egg interactions. Egg envelopes analyzed by SDS-PAGE were found to have almost identical gel patterns, whereas jelly component profiles were similar only for the larger macromolecules (>90 kDa). The cDNA sequences for egg envelope glycoproteins ZPA, ZPB, ZPC, ZPD and ZPAX, and also egg cortical granule lectin involved in the block to polyspermy, were cloned for X. tropicalis and showed a consistent ∼85% amino acid identity to the X. laevis sequences. Thus, homologous egg extracellular matrix molecules perform the same functions, and the molecular and cellular mechanisms of fertilization in these two species are probably equivalent.

Proteolytic processing of human zona pellucida proteins.

Formation of the egg's extracellular matrix, the zona pellucida, is critical for fertilization and development of growing embryos. Zona pellucida glycoproteins, ZP1, ZP2, and ZP3, are secreted to form an insoluble extracellular matrix surrounding mammalian eggs. All cloned mammalian zona pellucida sequences contain a furin consensus cleavage site, RX(K)/(R)R, upstream of a putative transmembrane domain, which suggests processing by an endoprotease of the furin-proprotein-convertase family. Recombinant expression of human (h) ZP1, ZP2, and ZP3 produces glycoproteins that are secreted and have migration patterns in SDS-PAGE identical to those of native human zona pellucida proteins. Because a C-terminal epitope tag that is present in the cell-associated zona proteins is, however, absent from the secreted zona proteins, secreted recombinant zona pellucida proteins lack their C-terminal regions. Three different strategies were used to explore processing events in the C-terminal region: site-directed mutagenesis of the furin cleavage site, treatment with a competitive inhibitor of all furin family members, and interference with Golgi modifications by Brefeldin A. All treatments altered the SDS-PAGE migration of recombinant hZP3, concordant with cleavage by a furin family member and Golgi glycosylation of secreted hZP3. Furthermore, cleavage of cell-associated hZP3 by exogenous furin converts the migration of cell-associated hZP3 to that of secreted hZP3. To determine whether a similar cleavage pattern exists in zona pellucida proteins that are assembled in the zona matrix, "hZP3 rescue" mouse zonae pellucidae were employed. Immunoblotting experiments revealed that hZP3, assembled and functional in the "hZP3 rescue" mouse zona pellucida, lacks the furin cleavage site, supporting the hypothesis that formation of the zona pellucida matrix involves regulated proteolysis by a member of the furin convertase family.

Synaptotagmin VIII Is Localized to the Mouse Sperm Head and May Function in Acrosomal Exocytosis1

The acrosome is a large secretory granule that undergoes exocytosis when receptors on the sperm surface bind ligands in the egg extracellular matrix. Acrosomal exocytosis resembles stimulated secretion in neurons in that it is triggered by a rise in intracellular Ca(2+). Synaptotagmins (Syt) comprise proteins thought to transduce this Ca(2+) signal to the fusion machinery. In this study, we showed that Syt VIII is present in spermatogenic cDNA libraries. Antiserum raised against a Syt VIII-specific peptide, which recognizes Syt VIII but does not cross-react with other Syt isoforms, labeled a single prominent band on Western immunoblots of mouse sperm homogenate. Syt VIII was restricted to the sperm membrane fraction enriched in markers associated with the mouse sperm head. Fluorescent immunocytochemistry on intact mouse sperm showed that Syt VIII is localized to the acrosomal crescent and is lost upon acrosome reaction. Moreover, the amount of Syt VIII remaining with the sperm decreased proportionately with the extent of acrosome-reacted sperm. Thus, Syt VIII is a candidate for the Ca(2+) sensor that regulates acrosomal exocytosis in mammalian sperm.

Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3.

In many cells, receptor activation initiates sustained Ca2+ entry which is critical in signal transduction. Mammalian transient receptor potential (Trp) proteins, which are homologous to the Drosophila photoreceptor-cell Trp protein, have emerged as candidate subunits of the ion channels that mediate this influx. As a consequence of overexpression, these proteins produce cation currents that open either after depletion of internal Ca2+ stores or through receptor activation. However, determining the role of endogenous Trp proteins in signal transduction is complicated by the absence of selective antagonists. Here we examine Trp function during sperm-egg interaction. The sperm acrosome reaction is a Ca2+-dependent secretory event that must be completed before fertilization. In mammals, exocytosis is triggered during gamete contact by ZP3, a glycoprotein constituent of the egg's extracellular matrix, or zona pellucida (ZP). ZP3 activates trimeric G proteins and phospholipase C and causes a transient Ca2+ influx into sperm through T-type Ca2+ channels. These early responses promote a second Ca2+-entry pathway, thereby producing sustained increases in intracellular Ca2+ concentration ([Ca2+]i) that drive acrosome reactions. Our results show that Trp2 is essential for the activation of sustained Ca2+ influx into sperm by ZP3.